RESUMEN
The dinitrogenase reductase gene (nifH) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field.IMPORTANCE Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.
Asunto(s)
Bacterias/genética , Microbiota/genética , Fijación del Nitrógeno , Oxidorreductasas/genética , Microbiología del Suelo , Bacterias/clasificación , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Biología Computacional , ADN Bacteriano/genética , Ecosistema , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenómica , Microbiota/fisiología , Nitrógeno/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa , Rizosfera , Análisis de Secuencia de ADNRESUMEN
Previous studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.
Asunto(s)
Proteínas Bacterianas/genética , Genoma Bacteriano , Bacterias Fijadoras de Nitrógeno/fisiología , Saccharum/microbiología , Agricultura , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Genómica/métodos , Klebsiella/genética , Klebsiella/aislamiento & purificación , Bacterias Fijadoras de Nitrógeno/efectos de los fármacos , Bacterias Fijadoras de Nitrógeno/genética , Bacterias Fijadoras de Nitrógeno/aislamiento & purificación , Oxidorreductasas/genética , Rizosfera , Microbiología del Suelo , Factores de Virulencia/genéticaRESUMEN
Members of the Klebsiella genus promote plant growth. We report here draft whole-genome sequences for 15 Klebsiella sp. isolates from sugarcane fields in the Cauca Valley of Colombia. The genomes of these isolates were characterized as part of a broader effort to evaluate their utility as endemic plant growth-promoting biofertilizers.